The structural changes exhibited by the small protein, calmodulin, upon sequential binding of Ca ion are to be monitored in terms of volume changes, both equilibrium and hydrodynamic. The putatively smaller conformational changes occurring upon interaction with Mg ion and those resulting from competitive binding via mixtures of Ca ion and Mg ion with calmodulin are also to be reflected by volume change behavior. Since calmodulin participates in the regulation of many cellular processes, it is necessary to determine if multiple, stable conformations exist as a test of the multiple recognition sites hypothesis (e.g., transient Mg ion fluxes in the cell may cause multiple changes in the structural patterns of calmodulin-Ca ion complexes). The equilibrium volume change, delta V, is a sensitive indicator of chemical change. Although the relation of delta V to individual chemical events in protein interactions is not completely understood, further insights will be gained by determining the change in partial volume of all components (protein, water, metal ions) in the system as a function of ion concentration. In particular, the difference in partial volume of an ion in the presence and absence of protein has been found to be a sensitive indicator of strong versus weak interactions. This thermodynamic study will be carried out by a densimetric procedure utilizing a rapid magnetic densimeter with precisions in delta V of plus or minus 5 nanoliters. The change in hydrodynamic volume of calmodulin as a function of Ca ion, Mg ion and their mixtures is also proposed. This will be accomplished with a sensitive magnetic viscometer equipped with kinetic capabilities and computer-assisted measurement and data processing; hence, slow structural changes of the complexes as a result of metal ion binding can be monitored. This study may help explain why calmodulin migrates as a larger protein on gel columns and exhibits a different second virial coefficient by sedimentation analysis in the presence and absence of Ca ion. The volume properties by both methods will be compared and interpreted in connection with measurements from different probes reported by other investigators.